Skip to main content
NCBI home page
iScience logoLink to iScience
. 2020 Jan 14;23(2):100838. doi: 10.1016/j.isci.2020.100838

DNMT3B Oncogenic Activity in Human Intestinal Cancer Is Not Linked to CIMP or BRAFV600E Mutation

Douglas J MacKenzie 1, Neil A Robertson 1, Iqbal Rather 1,5, Claire Reid 1, Gintare Sendzikaite 1, Hazel Cruickshanks 1, Tony McBryan 1, Andrew Hodges 4, Catrin Pritchard 2, Karen Blyth 1,3, Peter D Adams 1,4,6,∗
PMCID: PMC7000804  PMID: 32058953

Summary

Approximately 10% of human colorectal cancer (CRC) are associated with activated BRAFV600E mutation, typically in absence of APC mutation and often associated with a CpG island methylator (CIMP) phenotype. To protect from cancer, normal intestinal epithelial cells respond to oncogenic BRAFV600E by activation of intrinsic p53 and p16-dependent tumor suppressor mechanisms, such as cellular senescence. Conversely, CIMP is thought to contribute to bypass of these tumor suppressor mechanisms, e.g. via epigenetic silencing of tumor suppressor genes, such as p16. It has been repeatedly proposed that DNMT3B is responsible for BRAFV600E-induced CIMP in human CRC. Here we set out to test this by in silico, in vitro, and in vivo approaches. We conclude that although both BRAFV600E and DNMT3B harbor oncogenic potential in vitro and in vivo and show some evidence of cooperation in tumor promotion, they do not frequently cooperate to promote CIMP and human intestinal cancer.

Subject Areas: Biological Sciences, Molecular Biology, Cancer

Graphical Abstract

graphic file with name fx1.jpg

Open in a new tab

Highlights

  • •

    DNMT3B antagonizes BRAFV600E-induced senescence-like state

  • •

    DNMT3B accelerates BRAFV600E-induced intestinal cancer

  • •

    Other studies do not support a role for DNMT3B in CIMP and cooperation with BRAFV600E

  • •

    On balance, BRAFV600E and DNMT3B are unlikely to cooperate in human intestinal cancer

Biological Sciences; Molecular Biology; Cancer

Introduction

In normal cells, the BRAF kinase, encoded by the BRAF gene, is a critical effector of cell signaling pathways, most notably the RAS-BRAF-MEK-ERK mitogenic pathway. This proto-oncogenic pathway is activated by genetic mutations at some point along the cascade in most human cancers (Yaeger and Corcoran, 2019). Approximately 10% of human CRC are associated with activated BRAFV600E mutation. Although inhibitors of activated BRAF, such as Vemurafenib, are of some benefit in other cancers harboring BRAFV600E, such as melanoma, in CRC these inhibitors are of limited therapeutic value (Dienstmann et al., 2017). Yet CRC harboring BRAFV600E has a poor prognosis (Samowitz et al., 2005b, Ogino et al., 2009). Thus, it is important to understand the oncogenic mechanisms underlying this disease.

Activated BRAFV600E mutation is typically found in absence of mutation in the Adenomatous Polyposis Coli (APC) gene, a gene that is mutated and inactivated in ∼80% of CRC (Dienstmann et al., 2017). Activated BRAFV600E drives hyperproliferation and neoplasia via the MEK-ERK mitogenic pathway (Lavoie and Therrien, 2015). However, to protect from cancer, normal intestinal epithelial cells are thought to respond to oncogenic BRAFV600E by activation of intrinsic TP53 (p53) and CDKN2A (p16)-dependent tumor suppressor mechanisms (Rad et al., 2013), including cellular senescence (Carragher et al., 2010, Kriegl et al., 2011). In response to acquisition of an activated oncogene, primary human cells can enter a proliferation-arrested senescent state (oncogene-induced senescence [OIS]) that suppresses tumor formation (Michaloglou et al., 2005, Chen et al., 2005, Braig et al., 2005, Collado et al., 2005, He and Sharpless, 2017). Senescent cells also exhibit an altered secretory program, the so-called Senescence Associated Secretory Phenotype (SASP) (Kuilman et al., 2008, Acosta et al., 2008, Krtolica et al., 2001) comprised of pro-inflammatory cytokines and chemokines, which also contributes to tumor suppression by promoting clearance of senescent cells by the immune system (Lujambio et al., 2013, Xue et al., 2007, Kang et al., 2011).

Conversely, CpG island methylator phenotype (CIMP) is thought to contribute to bypass of tumor suppressor mechanisms by epigenetic silencing of tumor suppressor genes, such as MLH1 and CDKN2A (Lao and Grady, 2011). In CRC, BRAFV600E mutation and CIMP are quite tightly linked (Ogino et al., 2009, Weisenberger et al., 2006, Inoue et al., 2015, Nagasaka et al., 2004, Nagasaka et al., 2008, Hinoue et al., 2012, Cancer Genome Atlas Network, 2012), leading to speculation that BRAFV600E promotes and/or selects for CIMP en route to tumor progression. Because CDKN2A encodes p16, a key effector of cellular senescence (He and Sharpless, 2017), CIMP might be expected to suppress OIS.

DNA methyl transferase 3B (DNMT3B) is a so-called de novo DNA methyl transferase, capable of methylating CpGs where both CpGs of the palindrome are in the unmethylated state. In normal mouse intestine, inactivation of Dnmtb does not have a marked phenotype (Elliott et al., 2016, Lin et al., 2006). However, several studies have suggested that elevated DNMT3B might cooperate with BRAFV600E to drive tumorigenesis and that DNMT3B is responsible for methylation of DNA to generate CIMP (Carragher et al., 2010, Fang et al., 2014, Ibrahim et al., 2011, Nosho et al., 2009). According to one model in which the link between oncogene activation and CIMP can be quite indirect, oncogene-induced senescence and/or other oncogene-activated p53 and p16-dependent tumor suppressor mechanisms provide the selective pressure for DNMT3B and CIMP-mediated silencing of these key tumor suppressor pathways. Consistent with this model, in a mouse model of BrafV600E-driven colon cancer, escape from senescence and tumor progression was linked to increased expression of Dnmt3b and methylation and silencing of p16 (Carragher et al., 2010). According to this model, although activated BRAFV600E can increase the selective pressure that favors CIMP, methylation is not directly caused by BRAFV600E but has an inherent tendency to encroach on unmethylated CpG islands during aging (Skvortsova et al., 2019, Ushijima and Suzuki, 2019, Tao et al., 2019). In another model that more directly links oncogenic signaling and CIMP, it has been proposed that BRAFV600E signaling recruits DNMT3B to genes silenced by CIMP via the transcriptional repressor, MAFG, thereby directly promoting CIMP (Fang et al., 2014, Fang et al., 2016).

However, studies into the relationship between BRAFV600E and DNMT3B to this point are incomplete. For example, their associations in human TCGA data and functional interactions in mouse models have not, as far as we are aware, been considered in detail in previous studies. Here we set out to investigate whether BRAFV600E and DNMT3B cooperate in intestinal tumorigenesis by examination of human tumor data, in vitro models of senescence, and mouse models of CRC. The results present a mixed story, but are important for the field, and, on balance, lead us to suggest that BRAFV600E mutation and DNMT3B do not frequently cooperate to promote CIMP and human intestinal cancer.

Results

DNMT3B Is Frequently Amplified and Overexpressed in Human CRC

To begin to probe the putative oncogenic function of DNMT3B in human CRC, we mined human TCGA data examining levels of DNMT3B expression and gene copy number. First, we confirmed that, at the mRNA level, DNMT3B is expressed at a higher level in human CRC than normal human colon (Figures 1A and S1A–S1C). We also found that the DNMT3B gene exhibits increased copy number, indicative of gene amplification, in a substantial proportion of CRC (Figure 1B). Amplification of DNMT3B in human CRC is also apparent via Database: cbioportal.org (Gao et al., 2013). Moreover, comparing DNMT3B mRNA expression and gene copy number revealed a correlation between DNMT3B copy number and mRNA expression (Figures 1C and 1D). These data are consistent with the idea that increased DNMT3B expression can be oncogenic and suggest that increased expression in human CRC is often a result of gene amplification.

Figure 1.

Figure 1

Open in a new tab

DNMT3B Is Frequently Amplified and Overexpressed in CRC

(A) Violin plot showing distribution of DNMT3B expression levels in 207 CRC tumor samples. Mann-Whitney U test; p value = 5.785e-10.

(B) DNMT3B relative copy number across 207 tumor samples. Lines at y axis −0.2 and 0.2 indicate the threshold for deletion and amplification, respectively.

(C) Median centered log2 DNMT3B mRNA expression plot from 207 tumor samples (TCGA) annotated with BRAF mutation status.

(D) The relationship between DNMT3B relative copy number and DNMT3B mRNA expression in 207 tumor samples.

Ectopic Expression of DNMT3B Antagonizes BRAFV600E-Induced Proliferation Arrest and SASP

Previous studies suggested that elevated DNMT3B might cooperate with BRAFV600E to drive tumorigenesis (Carragher et al., 2010, Fang et al., 2014, Ibrahim et al., 2011, Nosho et al., 2009). To investigate this possibility of oncogenic cooperation between BRAFV600E and elevated DNMT3B, we tested functional interactions between the two in an in vitro cell culture model. In the absence of a robust normal human colonic epithelial cell model, we turned to primary human fibroblasts that were previously used to model molecular events underlying CIMP in CRC (Fang et al., 2014) and more generally have been a paradigm for investigating mechanisms of oncogene cooperation in human cells (Hahn et al., 1999, Hahn et al., 2002). Primary human IMR90 fibroblasts were infected with drug-selectable lentiviruses directing expression of BRAFV600E, DNMT3B, or empty vector as a control. Both proteins were efficiently expressed for up to 10 days post-infection (Figure 2A). When expressed on its own, activated BRAFV600E induced a marked decrease in the percent of cells in S phase of the cell cycle, in line with this oncogene's ability to induce senescence-associated proliferation arrest in primary cells (Michaloglou et al., 2005, Pawlikowski et al., 2013) (Figure S2A). The decline in cells in S phase was accompanied by decreased abundance of cell cycle drivers, cyclin A, and phosphorylated pRB, and, perhaps accounting for decreased phosphorylated pRB through regulation of p16INK4A (McHugh and Gil, 2018), decreased expression of EZH2 (Figure 2A). Ectopic expression of DNMT3B increased the proportion of cells in S phase, proportionately more so in cells ectopically expressing BRAFV600E (Figures 2B and S2A). In cells expressing both BRAFV600E and DNMT3B compared with those expressing BRAFV600E only, this was accompanied by increased cyclin A, EZH2 and phosphorylated pRB, and, at the seven-day time point, a consistent decrease in expression of cell cycle inhibitor, CDKN1B (p27KIP1) (Figure 2A). Expression of DNMT3B alone had no detectable effect on these key cell-cycle regulators (Figure 2A). We conclude that elevated DNMT3B antagonizes BRAFV600E-induced cell-cycle arrest.

Figure 2.

Figure 2

Open in a new tab

DNMT3B Suppresses Features of OIS

(A) Kinetic analysis assessing the impact of ectopic DNMT3B on markers of BRAFV600E-induced cell-cycle exit of primary IMR90 cells, determined by Western blot. MW markers in kDa.

(B) Impact of ectopic DNMT3B on BRAFV600E-induced S-phase exit of primary IMR90 cells, determined by BrdU labeling. Each group n = 4, from days 3, 5, 7, and 10 post-infection. Middle line = median; box top/bottom = upper and lower quartiles; whiskers 90th (upper) & 10th (lower) percentile of data. Kruskal-Wallis test, p < 0.05.

(C) Number of significantly differentially expressed genes between indicated pairwise comparisons of primary IMR90 cells expressing ectopic BRAFV600E and/or DNMT3B and/or empty vectors, determined from RNA-seq FPKM values (Cuffdiff, FDR corrected p value of <0.05).

(D) Number of significantly up or down genes between indicated pairwise comparisons of primary IMR90 cells expressing ectopic BRAFV600E and/or DNMT3B and/or empty vectors, determined from RNA-seq FPKM values.

(E) Heatmap of expression of significant changed proliferation-associated genes.

(F) Heatmap of expression of significant changed SASP genes.

To better understand the effect of DNMT3B on BRAFV600E-induced cell-cycle arrest, we infected cells with empty vector, DNMT3B alone, BRAFV600E alone, or the combination and performed RNA-seq. Principal component analysis showed that three replicates of each condition expressed distinct transcriptomes (Figure S2B). Expression of activated BRAFV600E significantly increased and decreased expression of 3403 and 5745 genes, respectively (Figures 2C and 2D). These changes in gene expression include those characteristic of OIS, including repression of cell cycle genes and activation of inflammatory genes indicative of the SASP (Figures 2E and 2F). However, co-expression of DNMT3B antagonized a substantial proportion of the gene expression changes induced by BRAFV600E (Figure 2D) (specifically, 1,055 of genes upregulated by BRAFV600E and 2,546 of genes downregulated by BRAFV600E). Consistent with the previous cell-cycle analyses (Figures 2A, 2B, and S2A), DNMT3B modestly suppressed BRAFV600E-induced repression of many cell proliferation genes (Figures 2E, S2C, and S2D). In addition, DNMT3B markedly suppressed many of the SASP genes induced by BRAFV600E (Figure 2F). DNMT3B-mediated suppression of BRAFV600E-induced repression of cell-cycle genes and activation of SASP was also apparent by GSEA analysis (Figure S2E). In sum, elevated DNMT3B impaired two key tumor suppressive phenotypes induced by BRAFV600E in primary human cells, cell-cycle arrest and SASP. These data show that combined expression of BRAFV600E and DNMT3b could cooperate to promote tumorigenesis.

Dnmt3b Enhances Tumorigenesis and Decreases Survival in a BrafV600E-Driven Mouse Model of Intestinal Cancer

To investigate the tumor promoting potential of combined BrafV600E mutation and elevated Dnmt3b in vivo, we tested the two in an in vivo mouse model of intestinal tumorigenesis. A previous study reported that mouse intestinal-specific expression of activated Braf under control of a constitutive villin-Cre recapitulates features of the human serrated pathway of intestinal tumorigenesis (Rad et al., 2013). In an effort to better mimic cancer initiation in human adult tissues, in a modified version of this model, mice expressing a conditional-activated intestinal-specific BrafV600E oncogene under the control of tamoxifen-inducible Villin-Cre-ERT2 were crossed to mice expressing conditional Dnmt3b under control of doxycycline inducible rtTA (el Marjou et al., 2004, Linhart et al., 2007, Steine et al., 2011). Transgene expression was induced in 6- to 10-week-old mice, and western blotting confirmed inducible expression of Dnmt3b in the small intestine and colon (Figure 3A). Mice were culled when they showed comparable clinical signs of disease. Accordingly, at the time the BrafV600E and BrafV600E/Dnmt3b mice were culled, the two groups harbored a comparable number of small intestinal tumors (Figures 3B and 3C); no overt differences in tumor histology were noted between BrafV600E and BrafV600E/Dnmt3b tumors. No colonic tumors were identifiable in any mice examined. At this time, we did not detect any overt differences between proliferation and senescence markers in BrafV600E/Dnmt3b and BrafV600E mice, in either tumor or histologically normal intestine (Figures 3D, 3E, and S3A and data not shown). Strikingly, however, combined ectopic Dnmt3b expression together with activated BrafV600E in the murine intestine resulted in a marked decrease in overall survival compared with BrafV600E alone (Figure 3F). Median post-survival induction in mice expressing BrafV600E and endogenous Dnmt3b was 489 days, compared with 289 in mice with ectopic BrafV600E and Dnmt3b. Mice expressing ectopic Dnmt3b alone did not develop any clinical signs of illness up until 377 days following induction and were culled at this time. Neither intestinal abnormalities nor tumors were identified in these mice, consistent with previous reports (Steine et al., 2011, Linhart et al., 2007). These results suggest that elevated Dnmt3b accelerated the accumulation of a lethal burden of BrafV600E-driven intestinal tumors.

Figure 3.

Figure 3

Open in a new tab

DNMT3B Accelerates Intestinal Cancer

(A) Western blot showing doxycycline-inducible expression of Dnmt3b in small intestine (S) and colon (C) of R26-M2-rtTA;Col1a1-tetO-Dnmt3b1 and R26-M2-rtTA;WT mice. MW markers in kDa.

(B) Number of small intestine tumors in VilCreErT2;LSL BrafV600E mice with and without ectopic Dnmt3b expression. Mean with error bars −/+ SEM. Mann Whitney test p = 0.98 (ns) n = 5 vs 5.

(C) Representative small intestine tumor fromVilCreErT2;LSL BrafV600E mice, similar with or without Dnmt3b.

(D) Representative immunohistochemistry images for Ki67 in VilCreErT2; LSLBrafV600E mice with and without ectopic Dnmt3b. Scale bars are 200 μM.

(E) Representative immunohistochemistry images for p21 in VilCreErT2;LSLBrafV600E mice with and without ectopic Dnmt3b. Scale bars are 200 μM.

(F) Kaplan-Meier curve comparing survival in VilCreErT2; LSLBrafV600E mice with and without intestinal Dnmt3b ectopic expression. “DNMT3B only” mice (“Col1a1-tetO-Dnmt3b1 +/−”) also shown. Log rank (Mantel-Cox) test, p < 0.0001.

(G) Total small intestine tumor number in VilCreErT2; LSLBrafV600E; Dnmt3bwt/wt and VilCreErT2; LSLBrafV600E; Dnmt3bfl/fl mice. Mann Whitney test p = 0.42; n = 5 vs 5. Mean with error bars −/+ SEM.

(H) Kaplan-Meier curve of survival in VilCreErT2;LSLBrafV600E mice with wild-type or floxed Dnmt3b. Log Rank (Mantel-Cox) test, p < 0.02.

To test whether Dnmt3b is required for BrafV600E-induced intestinal tumorigenesis, we generated mice harboring a conditionally activated BrafV600E oncogene and conditionally inactivated Dnmt3bfl/fl, both under control of Villin-Cre-ERT2 (Figure S3B). Similar to the observations with elevated Dnmt3b, inactivation of Dnmt3b did not significantly affect the number of intestinal tumors at the time of terminal disease (Figure 3G). Again, no differences in tumor histology were noted either. However, inactivation of Dnmt3b did significantly extend survival of mice harboring BrafV600E (Figure 3H). Consistent with the previous elevated Dnmt3b model, these results suggest that abundance of Dnmt3b is rate-limiting for acquisition of a lethal burden of tumors in this BrafV600E-induced intestinal cancer model. Overall, these data support the notion that the combination of BrafV600E and elevated Dnmt3b can be potently oncogenic in CRC.

Activated BRAFV600E Repressed Expression of DNMT3b and Failed to Induce a CIMP Phenotype

According to a previously proposed model, activated BRAFV600E signaling recruits DNMT3B to genes silenced by CIMP via the transcriptional repressor, MAFG (Fang et al., 2014, Fang et al., 2016). This model was based, in part, on the ability of BRAFV600E to increase expression of MAFG and decrease expression of CIMP target gene MLH1 in primary human fibroblasts (Fang et al., 2014). To extend this line of investigation, we ectopically expressed activated BRAFV600E in primary human fibroblasts and assessed its impact on DNMT3B and CIMP. As shown previously (Michaloglou et al., 2005), ectopic expression of BRAFV600E induced proliferation arrest and upregulation of markers of senescence (Figures 2A and 4A). However, surprisingly, activated BRAFV600E triggered downregulation of DNMT3B expression (Figure 4A). Next, we set out to ask whether oncogenic BRAFV600E is able to induce methylation changes characteristic of CIMP. Primary human fibroblasts were again infected with a lentivirus directing expression of BRAFV600E. Seven days after infection, the cells were harvested, DNA prepared, and subject to WGBS to map the DNA methylome. Principal Component Analysis poorly separated the control and BRAFV600E-expressing cells (Figure S4A). Consistent with this, although we reported previously that replicative senescent (RS) cells undergo a marked global hypomethylation (Cruickshanks et al., 2013), BRAFV600E-expressing cells showed only a very small overall gain in methylation (Figure S4B). A scatterplot of CpG methylation in control versus BRAFV600E-expressing cells also revealed no substantial differences, in contrast to a comparison of proliferating and replicative senescent fibroblasts, which revealed marked hypomethylation in the latter (Figure 4B) (Cruickshanks et al., 2013). The relative similarity of vector and BRAFV600E cells, compared with proliferating versus replicative senescent, was underscored by whole chromosome difference plot (Figure S4B). This revealed the marked hypomethylation of lamin-associated domains (LADs) in replicative senescent cells reported previously (Cruickshanks et al., 2013), but this was absent from BRAFV600E cells (Figure S4B). Indeed, although many significantly differentially methylated individual CpGs and regions (DMRs) were detected between proliferating and RS cells, very few were detected between control and BRAFV600E-expressing cells (Figure S4C). Although increasing the window size of candidate DMRs decreased the number of DMRs detected in replicative senescent cells due to merging of small DMRs, varying the size of candidate DMRs did not majorly affect the number of DMRs detected in BRAFV600E cells (Figure S4C). Most relevant to a candidate CIMP-like phenotype, BRAFV600E-expressing cells did not gain methylation at CpG islands whose methylation is diagnostic of CIMP, including MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1; this is in contrast to replicative senescent cells (Figure 4C) (Cruickshanks et al., 2013). Taken together, these results do not support a role for BRAFV600E in direct induction of CIMP via DNMT3B, at least in fibroblasts.

Figure 4.

Figure 4

Open in a new tab

Ectopic Expression of DNMT3B Does Not Induce CIMP

(A) Western blot of lysates from human fetal lung fibroblasts (IMR-90) infected with lentivirus directing expression of BRAFV600E or corresponding empty vector. MW markers in kDa.

(B) Scatterplot of CpG methylation in BRAFV600E-infected fibroblasts compared with proliferating “vector” controls (top), and replicative senescent (PD88) compared with proliferating controls (PD28) (bottom).

(C) Smoothened methylation plots of CIMP panel genes in vector and BRAFV600E-expressing cells. Red and blue lines indicate the smoothened average percentage methylation at corresponding CpGs. Individual CpGs are indicated by black “ticks” along the x axis.

BRAFV600E Mutation Is Neither Necessary nor Sufficient for CIMP, and BRAFV600E Mutations and CIMP Are Both Linked to a Low Expression of DNMT3B

In light of these functional data, we turned again to human TCGA data to shed more light on the potential interactions between BRAFV600E and DNMT3B in human CRC. First, we clustered patient samples based on methylation values of all probes at CpG islands showing significant variance across the human TCGA CRC datasets. As expected, this separated out human CRC into at least three groups, one of relatively high methylation at CpG islands, CIMP high (CIMP-H), and CIMP low (CIMP-L) and non-CIMP (Hinoue et al., 2012, Cancer Genome Atlas Network, 2012) (Figure 5A). As expected, and as reported previously (Hinoue et al., 2012, Cancer Genome Atlas Network, 2012, Weisenberger et al., 2006, Samowitz et al., 2005a), CIMP-H tumors associated with BRAFV600E mutation (Figure 5A) (2.16-fold enrichment, p < 0.001). Somewhat surprisingly, however, CIMP-H tumors were anti-correlated with DNMT3B copy number amplification (Figure 5A) (0.21-fold enrichment, p < 0.001). The same relationship was apparent when we analyzed only samples with microsatellite instability (MSI+), known to be enriched in CIMP (Guinney et al., 2015) (Figure S5A). To examine more closely the relationship between CIMP and DNMT3B amplification, we rank-ordered all human CRC samples by the degree of DNMT3B amplification (which correlates with expression (Figures 1C and 1D)) and compared with CIMP. Amplification of DNMT3B was not associated with CIMP and, indeed, the tumors with the most overt CIMP appeared to associate with relatively low DNMT3B copy number (Figure 5B). Next, we directly compared CIMP score with DNMT3B amplification or expression. This confirmed an inverse relationship between CIMP and both DNMT3B amplification and expression (Figures 5C, 5D, and S5A). Finally, we directly compared DNMT3B expression and BRAFV600E mutation. This showed that DNMT3B expression is higher in those tumors with wild-type BRAF (Figures 5E, S5C, and S5D). In sum, this analysis of human TCGA confirms the strong association between BRAF mutation and CIMP-H but does not support a marked association between amplification and increased expression of DNMT3B and either BRAF mutation or CIMP-H and so challenges the idea that elevated DNMT3B contributes to BRAFV600E-induced tumorigenesis and associated CIMP in humans.

Figure 5.

Figure 5

Open in a new tab

Human BRAFV600E/CIMP Tumors Express Low Levels of DNMT3B

(A) Clustering of TCGA human CRC based on Illumina 450K DNA methylation array data (all CpGs showing variation >0.2 StdDev). BRAF mutation and DNMT3B amplification status are shown below (red = mutated/amplified).

(B) CpG probes (y axis of the heatmap) clustered based on increasing DNMT3B copy number (x axis of the heatmap).

(C) Relationship between DNMT3B copy number and methylation of CIMP biomarker genes (CACNA1G, RUNX3, IGF2, MLH1, NEUROG1, CRABP1, CDKN2A). Red line marks the boundary between non-amplified and amplified DNMT3B. Each black dot indicates the mean methylation score in the indicated probes in one patient sample. Y axis—mean methylation, x axis—relative copy number.

(D) Boxplot of DNMT3B FPKM mRNA expression in human colorectal carcinoma TCGA patients subdivided by CIMP status. Mann Whitney test, p = 0.001.

(E) Violin plot showing distribution of DNMT3B expression levels in 207 CRC tumor samples, separated according to BRAF WT or BRAFV600E mutant. p value = 0.0001.

Discussion

Approximately 10% of human colorectal cancers harbor a BRAFV600E mutation, which has been demonstrated to act as a founder mutation for an alternative serrated pathway of colorectal carcinogenesis (Cancer Genome Atlas Network, 2012, Fransen et al., 2004, Vaughn et al., 2011, Giannakis et al., 2016, Carragher et al., 2010, Rad et al., 2013). However, on its own, both in vitro and in vivo, activated oncogenic BRAFV600E induces OIS, an established tumour-suppressive mechanism (Michaloglou et al., 2005, Carragher et al., 2010, Rad et al., 2013, Dankort et al., 2007). Consistent with a potent BRAFV600E-induced tumor suppressor mechanism in humans, activating BRAFV600E mutations are detectable in 62%–70% of hyperplastic colonic polyps: lesions traditionally considered to harbor no oncogenic potential (Yang et al., 2004, Mesteri et al., 2014). Furthermore, the existence of an OIS barrier in human serrated pathway carcinogenesis is supported by the published in situ data (Kriegl et al., 2011).

It is clear, therefore, that additional genetic and/or epigenetic events are required to promote neoplastic transformation in the serrated pathway, which if untreated (e.g. by endoscopic polypectomy) eventually culminates in the development of invasive carcinoma. CIMP is thought to promote tumorigenesis by bypass of tumour-suppressor mechanisms, such as silencing of CDKN2A/INK4A (Kriegl et al., 2011, Carragher et al., 2010). It has been recognized for some time that there is an extremely close relationship between BRAFV600E mutations and CIMP-H in human colorectal cancer (Ogino et al., 2009, Weisenberger et al., 2006, Inoue et al., 2015, Nagasaka et al., 2004, Nagasaka et al., 2008, Hinoue et al., 2012, Cancer Genome Atlas Network, 2012). Although this association was initially correlative, it has recently been proposed that BRAFV600E can directly induce CIMP through the de novo methyltransferase, DNMT3B (Fang et al., 2014, Fang et al., 2016). Separately, elevated DNMT3B expression has previously been linked to the development of CIMP in both murine and human colorectal neoplasia (Carragher et al., 2010, Ibrahim et al., 2011, Nosho et al., 2009, Steine et al., 2011). Furthermore, DNMT3B has been demonstrated to have an oncogenic function in murine colon cancer (Lin et al., 2006, Linhart et al., 2007). Thus, the emerging dogma is of a model in which neoplastic transformation in BRAFV600E-mutant colorectal serrated lesions may be contributed to by the ability of this oncogene to induce a CpG island methylator phenotype, mediated by DNMT3B.

There are, however, several obvious paradoxes. Firstly, only a minority of colorectal lesions harboring an activating BRAFV600E mutation eventually progress to invasive carcinoma (Yang et al., 2004, Mesteri et al., 2014). Secondly, CIMP develops gradually with serrated pathway progression (Inoue et al., 2015). Thirdly, as many as 40% of CIMP-H tumors do not harbor an activating BRAFV600E mutation. Finally, approximately 10% of patients with an activating BRAFV600E mutation are CIMP-L or CIMP-negative (Ogino et al., 2009). Thus, a simple, linear model by which BRAFV600E directly induces CIMP cannot be fully reconciled with the disease biology. We therefore set out to comprehensively examine the relationships between an activated BRAFV600E oncogene, DNMT3B, and CIMP in colorectal cancer by multiple approaches.

Because activated BRAFV600E is a potent inducer of OIS (Michaloglou et al., 2005, Carragher et al., 2010, Rad et al., 2013, Dankort et al., 2007), we initially set out to test the idea that elevated DNMT3B can bypass BRAFV600E-induced OIS. Indeed, ectopic expression of DNMT3B together with BRAFV600E partially suppressed two phenotypes characteristic of BRAFV600E-induced OIS, namely proliferation arrest and, most notably, SASP. Extending these results in vivo, conditional inducible and knockout mouse models showed that expression of Dnmt3b is rate-limiting for accumulation of a lethal burden of BrafV600E-driven tumors in mouse intestinal epithelium. The molecular mechanisms underlying these phenotypes remain to be established. Conceivably, suppression of SASP gene expression by DNMT3B could depend on suppression of BRAFV600E signaling, de novo methylation of SASP genes, or another mechanism. Regardless of the specific mechanism, these in vitro and in vivo lines of evidence support an oncogenic function for DNMT3B in cooperation with BRAFV600E.

However, our other analyses do not support a role for DNMT3B cooperating with activated BRAFV600E via its induction of CIMP. Since activated BRAFV600E has been proposed to directly potentiate DNMT3B-mediated CIMP and silencing of tumor suppressor genes (Fang et al., 2014, Fang et al., 2016), we wondered whether BRAFV600E-induced OIS might simultaneously sow the seeds of its own destruction, in the form of DNMT3B-mediated CIMP. However, in contrast to replicative senescent cells, in OIS BRAFV600E efficiently repressed expression of DNMT3B, and we observed very few BRAFV600E-induced DNA methylation changes. Finally, a closer examination of human TCGA data suggests that BRAFV600E and DNMT3B are unlikely to cooperate in tumorigenesis, and DNMT3B is unlikely to mediate CIMP. Analysis of data accessible through cBioportal, including TCGA and the largest study of 1,134 samples (Yaeger et al., 2018, Gao et al., 2013), also indicates that BRAFV600E mutation and DNMT3B amplification tend to be mutually exclusive. Taken together, these in vitro and in silico studies suggest that in BRAFV600E-driven CRC, elevated DNMT3B is likely not the cause of CIMP, and BRAFV600E and DNMT3B are unlikely to cooperate in progression of this disease. In summary, the data presented herein challenge the current model of the relationships between BRAF, DNMT3B, and CIMP in human colorectal cancer, whereby DNMT3B contributes to CIMP in association with BRAFV600E mutation and, in the most extreme form of the model, that mutant-BRAFV600E drives CIMP via DNMT3B. By multiple approaches, in vitro and by in silico analysis of TCGA, we have demonstrated that BRAFV600E mutation is associated with and can cause repression of DNMT3B. Furthermore, although DNMT3B overexpression and somatic copy number amplification are common features of human colorectal cancer, they are inversely correlated with CIMP. In functional in vitro studies, we obtained no evidence that DNMT3B is a driver of CIMP. Thus, although BRAFV600E and DNMT3B both harbor oncogenic potential, they do not appear to cooperate to induce CIMP and do not appear to cooperate frequently in human colorectal cancer by any mechanism.

Limitations of the Study

Of course, there are limitations to these conclusions drawn in part from “negative data” that we have been unable to address over the time frame of this study. First, although BRAFV600E does not induce detectable methylation changes characteristic of CIMP in a bulk population of cells, we cannot exclude the possibility that it does so in a very small subset of cells that is then prone to senescence escape, clonal outgrowth, and tumorigenesis. Even so, comparison of our WGBS data reported here for OIS with our previous studies in RS suggest that CIMP is more likely to arise in RS cells linked to other cumulative molecular stresses, e.g. telomere shortening, than in OIS cells (Cruickshanks et al., 2013). Second, our analysis of human TCGA data does not preclude an association between DNMT3B expression and BRAFV600E at very early stages of disease, prior to detection and inclusion of tumors in the TCGA study. Although these limitations may go some way toward reconciling our conclusions with those of previous studies, it is important to note that prior functional studies most directly linking DNMT3B to CIMP have been performed in established colon cancer cell lines in vitro (Fang et al., 2014). Despite these limitations to our study, we feel that, on balance, the weight of evidence does not support a role for DNMT3B and BRAFV600E cooperating in intestinal cancer.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

Work in the lab of PDA was supported by CRUK C10652/A16566; the lab of CP by CRUK program C1362/A13083; the lab of KB by Cancer Research UK (C596/A17196). Thanks to Owen Sansom for VilCreERT2 mice. We would like to thank the Core Services and Advanced to Technologies at the Cancer Research UK Beatson Institute (C596/A17196), with particular thanks to Biological Services Unit, Histology and Molecular Technologies. Additional support from Sanford Burnham Prebys NCI Cancer Center Support Grant P30 CA030199.

Author Contributions

D.J.M. performed the bulk of the experiments. I.R., C.R., and H.C. assisted with experiments. N.A.R., G.S., and T.M. performed computational data analysis. C.P. advised on experiments and generated the conditional BRAFV600E allele. K.B. advised on mouse models. A.H. in the SBP Bioinformatics Core provided statistical support. P.D.A. and D.M. conceived the study. P.D.A. wrote the manuscript. All authors edited and approved the manuscript.

Declaration of Interests

The authors have no competing interests to declare.

Published: February 21, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.100838.

Data and Code Availability

RNA-seq and DNA methylation data is available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126434. The accession number for the data reported in this paper is GSE126434: secure token for access exqpcwyshxsfdgd).

Supplemental Information

Document S1. Transparent Methods and Figures S1–S5
mmc1.pdf (3.4MB, pdf)

References

  1. Acosta J.C., O'loghlen A., Banito A., Guijarro M.V., Augert A., Raguz S., Fumagalli M., da Costa M., Brown C., Popov N. Chemokine signaling via the CXCR2 receptor reinforces senescence. Cell. 2008;133:1006–1018. doi: 10.1016/j.cell.2008.03.038. [DOI] [PubMed] [Google Scholar]
  2. Braig M., Lee S., Loddenkemper C., Rudolph C., Peters A.H., Schlegelberger B., Stein H., Dorken B., Jenuwein T., Schmitt C.A. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature. 2005;436:660–665. doi: 10.1038/nature03841. [DOI] [PubMed] [Google Scholar]
  3. Cancer Genome Atlas Network Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487:330–337. doi: 10.1038/nature11252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carragher L.A., Snell K.R., Giblett S.M., Aldridge V.S., Patel B., Cook S.J., Winton D.J., Marais R., Pritchard C.A. V600EBraf induces gastrointestinal crypt senescence and promotes tumour progression through enhanced CpG methylation of p16INK4a. EMBO Mol. Med. 2010;2:458–471. doi: 10.1002/emmm.201000099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen Z., Trotman L.C., Shaffer D., Lin H.K., Dotan Z.A., Niki M., Koutcher J.A., Scher H.I., Ludwig T., Gerald W. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature. 2005;436:725–730. doi: 10.1038/nature03918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Collado M., Gil J., Efeyan A., Guerra C., Schuhmacher A.J., Barradas M., Benguria A., Zaballos A., Flores J.M., Barbacid M. Tumour biology: senescence in premalignant tumours. Nature. 2005;436:642. doi: 10.1038/436642a. [DOI] [PubMed] [Google Scholar]
  7. Cruickshanks H.A., Mcbryan T., Nelson D.M., Vanderkraats N.D., Shah P.P., Van Tuyn J., Singh Rai T., Brock C., Donahue G., Dunican D.S. Senescent cells harbour features of the cancer epigenome. Nat. Cell Biol. 2013;15:1495–1506. doi: 10.1038/ncb2879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dankort D., Filenova E., Collado M., Serrano M., Jones K., Mcmahon M. A new mouse model to explore the initiation, progression, and therapy of BRAFV600E-induced lung tumors. Genes Dev. 2007;21:379–384. doi: 10.1101/gad.1516407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dienstmann R., Vermeulen L., Guinney J., Kopetz S., Tejpar S., Tabernero J. Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer. Nat. Rev. Cancer. 2017;17:79–92. doi: 10.1038/nrc.2016.126. [DOI] [PubMed] [Google Scholar]
  10. el Marjou F., Janssen K.P., Chang B.H., Li M., Hindie V., Chan L., Louvard D., Chambon P., Metzger D., Robine S. Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis. 2004;39:186–193. doi: 10.1002/gene.20042. [DOI] [PubMed] [Google Scholar]
  11. Fang M., Ou J., Hutchinson L., Green M.R. The BRAF oncoprotein functions through the transcriptional repressor MAFG to mediate the CpG Island Methylator phenotype. Mol. Cell. 2014;55:904–915. doi: 10.1016/j.molcel.2014.08.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elliott E.N., Sheaffer K.L., Kaestner K.H. The ‘de novo’ DNA methyltransferase Dnmt3b compensates the Dnmt1-deficient intestinal epithelium. Elife. 2016;5:e12975. doi: 10.7554/eLife.12975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fang M., Hutchinson L., Deng A., Green M.R. Common BRAF(V600E)-directed pathway mediates widespread epigenetic silencing in colorectal cancer and melanoma. Proc. Natl. Acad. Sci. U S A. 2016;113:1250–1255. doi: 10.1073/pnas.1525619113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fransen K., Klintenas M., Osterstrom A., Dimberg J., Monstein H.J., Soderkvist P. Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas. Carcinogenesis. 2004;25:527–533. doi: 10.1093/carcin/bgh049. [DOI] [PubMed] [Google Scholar]
  15. Gao J., Aksoy B.A., Dogrusoz U., Dresdner G., Gross B., Sumer S.O., Sun Y., Jacobsen A., Sinha R., Larsson E. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci. Signal. 2013;6:pl1. doi: 10.1126/scisignal.2004088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Giannakis M., Mu X.J., Shukla S.A., Qian Z.R., Cohen O., Nishihara R., Bahl S., Cao Y., Amin-Mansour A., Yamauchi M. Genomic correlates of immune-cell infiltrates in colorectal carcinoma. Cell Rep. 2016;15:857–865. doi: 10.1016/j.celrep.2016.03.075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guinney J., Dienstmann R., Wang X., de Reynies A., Schlicker A., Soneson C., Marisa L., Roepman P., Nyamundanda G., Angelino P. The consensus molecular subtypes of colorectal cancer. Nat. Med. 2015;21:1350–1356. doi: 10.1038/nm.3967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hahn W.C., Counter C.M., Lundberg A.S., Beijersbergen R.L., Brooks M.W., Weinberg R.A. Creation of human tumour cells with defined genetic elements. Nature. 1999;400:464–468. doi: 10.1038/22780. [DOI] [PubMed] [Google Scholar]
  19. Hahn W.C., Dessain S.K., Brooks M.W., King J.E., Elenbaas B., Sabatini D.M., Decaprio J.A., Weinberg R.A. Enumeration of the simian virus 40 early region elements necessary for human cell transformation. Mol. Cell Biol. 2002;22:2111–2123. doi: 10.1128/MCB.22.7.2111-2123.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. He S., Sharpless N.E. Senescence in health and disease. Cell. 2017;169:1000–1011. doi: 10.1016/j.cell.2017.05.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hinoue T., Weisenberger D.J., Lange C.P., Shen H., Byun H.M., van Den Berg D., Malik S., Pan F., Noushmehr H., van Dijk C.M. Genome-scale analysis of aberrant DNA methylation in colorectal cancer. Genome Res. 2012;22:271–282. doi: 10.1101/gr.117523.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ibrahim A.E., Arends M.J., Silva A.L., Wyllie A.H., Greger L., Ito Y., Vowler S.L., Huang T.H., Tavare S., Murrell A., Brenton J.D. Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression. Gut. 2011;60:499–508. doi: 10.1136/gut.2010.223602. [DOI] [PubMed] [Google Scholar]
  23. Inoue A., Okamoto K., Fujino Y., Nakagawa T., Muguruma N., Sannomiya K., Mitsui Y., Takaoka T., Kitamura S., Miyamoto H. B-RAF mutation and accumulated gene methylation in aberrant crypt foci (ACF), sessile serrated adenoma/polyp (SSA/P) and cancer in SSA/P. Br. J. Cancer. 2015;112:403–412. doi: 10.1038/bjc.2014.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kang T.W., Yevsa T., Woller N., Hoenicke L., Wuestefeld T., Dauch D., Hohmeyer A., Gereke M., Rudalska R., Potapova A. Senescence surveillance of pre-malignant hepatocytes limits liver cancer development. Nature. 2011;479:547–551. doi: 10.1038/nature10599. [DOI] [PubMed] [Google Scholar]
  25. Kriegl L., Neumann J., Vieth M., Greten F.R., Reu S., Jung A., Kirchner T. Up and downregulation of p16(Ink4a) expression in BRAF-mutated polyps/adenomas indicates a senescence barrier in the serrated route to colon cancer. Mod. Pathol. 2011;24:1015–1022. doi: 10.1038/modpathol.2011.43. [DOI] [PubMed] [Google Scholar]
  26. Krtolica A., Parrinello S., Lockett S., Desprez P.Y., Campisi J. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc. Natl. Acad. Sci. U S A. 2001;98:12072–12077. doi: 10.1073/pnas.211053698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kuilman T., Michaloglou C., Vredeveld L.C., Douma S., van Doorn R., Desmet C.J., Aarden L.A., Mooi W.J., Peeper D.S. Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network. Cell. 2008;133:1019–1031. doi: 10.1016/j.cell.2008.03.039. [DOI] [PubMed] [Google Scholar]
  28. Lao V.V., Grady W.M. Epigenetics and colorectal cancer. Nat. Rev. Gastroenterol. Hepatol. 2011;8:686–700. doi: 10.1038/nrgastro.2011.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lavoie H., Therrien M. Regulation of RAF protein kinases in ERK signalling. Nat. Rev. Mol. Cell Biol. 2015;16:281–298. doi: 10.1038/nrm3979. [DOI] [PubMed] [Google Scholar]
  30. Lin H., Yamada Y., Nguyen S., Linhart H., Jackson-Grusby L., Meissner A., Meletis K., Lo G., Jaenisch R. Suppression of intestinal neoplasia by deletion of Dnmt3b. Mol. Cell Biol. 2006;26:2976–2983. doi: 10.1128/MCB.26.8.2976-2983.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Linhart H.G., Lin H., Yamada Y., Moran E., Steine E.J., Gokhale S., Lo G., Cantu E., Ehrich M., He T. Dnmt3b promotes tumorigenesis in vivo by gene-specific de novo methylation and transcriptional silencing. Genes Dev. 2007;21:3110–3122. doi: 10.1101/gad.1594007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lujambio A., Akkari L., Simon J., Grace D., Tschaharganeh D.F., Bolden J.E., Zhao Z., Thapar V., Joyce J.A., Krizhanovsky V., Lowe S.W. Non-cell-autonomous tumor suppression by p53. Cell. 2013;153:449–460. doi: 10.1016/j.cell.2013.03.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McHugh D., Gil J. Senescence and aging: causes, consequences, and therapeutic avenues. J. Cell Biol. 2018;217:65–77. doi: 10.1083/jcb.201708092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mesteri I., Bayer G., Meyer J., Capper D., Schoppmann S.F., von Deimling A., Birner P. Improved molecular classification of serrated lesions of the colon by immunohistochemical detection of BRAF V600E. Mod. Pathol. 2014;27:135–144. doi: 10.1038/modpathol.2013.126. [DOI] [PubMed] [Google Scholar]
  35. Michaloglou C., Vredeveld L.C., Soengas M.S., Denoyelle C., Kuilman T., van Der Horst C.M., Majoor D.M., Shay J.W., Mooi W.J., Peeper D.S. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature. 2005;436:720–724. doi: 10.1038/nature03890. [DOI] [PubMed] [Google Scholar]
  36. Nagasaka T., Sasamoto H., Notohara K., Cullings H.M., Takeda M., Kimura K., Kambara T., Macphee D.G., Young J., Leggett B.A. Colorectal cancer with mutation in BRAF, KRAS, and wild-type with respect to both oncogenes showing different patterns of DNA methylation. J. Clin. Oncol. 2004;22:4584–4594. doi: 10.1200/JCO.2004.02.154. [DOI] [PubMed] [Google Scholar]
  37. Nagasaka T., Koi M., Kloor M., Gebert J., Vilkin A., Nishida N., Shin S.K., Sasamoto H., Tanaka N., Matsubara N. Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer. Gastroenterology. 2008;134:1950–1960.e1. doi: 10.1053/j.gastro.2008.02.094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nosho K., Shima K., Irahara N., Kure S., Baba Y., Kirkner G.J., Chen L., Gokhale S., Hazra A., Spiegelman D. DNMT3B expression might contribute to CpG island methylator phenotype in colorectal cancer. Clin. Cancer Res. 2009;15:3663–3671. doi: 10.1158/1078-0432.CCR-08-2383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ogino S., Nosho K., Kirkner G.J., Kawasaki T., Meyerhardt J.A., Loda M., Giovannucci E.L., Fuchs C.S. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut. 2009;58:90–96. doi: 10.1136/gut.2008.155473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pawlikowski J.S., Mcbryan T., van Tuyn J., Drotar M.E., Hewitt R.N., Maier A.B., King A., Blyth K., Wu H., Adams P.D. Wnt signaling potentiates nevogenesis. Proc. Natl. Acad. Sci. U S A. 2013;110:16009–16014. doi: 10.1073/pnas.1303491110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rad R., Cadinanos J., Rad L., Varela I., Strong A., Kriegl L., Constantino-Casas F., Eser S., Hieber M., Seidler B. A genetic progression model of Braf(V600E)-induced intestinal tumorigenesis reveals targets for therapeutic intervention. Cancer Cell. 2013;24:15–29. doi: 10.1016/j.ccr.2013.05.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Samowitz W.S., Albertsen H., Herrick J., Levin T.R., Sweeney C., Murtaugh M.A., Wolff R.K., Slattery M.L. Evaluation of a large, population-based sample supports a CpG island methylator phenotype in colon cancer. Gastroenterology. 2005;129:837–845. doi: 10.1053/j.gastro.2005.06.020. [DOI] [PubMed] [Google Scholar]
  43. Samowitz W.S., Sweeney C., Herrick J., Albertsen H., Levin T.R., Murtaugh M.A., Wolff R.K., Slattery M.L. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res. 2005;65:6063–6069. doi: 10.1158/0008-5472.CAN-05-0404. [DOI] [PubMed] [Google Scholar]
  44. Skvortsova K., Masle-Farquhar E., Luu P.L., Song J.Z., Qu W., Zotenko E., Gould C.M., Du Q., Peters T.J., Colino-Sanguino Y. DNA hypermethylation encroachment at CpG island borders in cancer is predisposed by H3K4 monomethylation patterns. Cancer Cell. 2019;35:297–314.e8. doi: 10.1016/j.ccell.2019.01.004. [DOI] [PubMed] [Google Scholar]
  45. Steine E.J., Ehrich M., Bell G.W., Raj A., Reddy S., van Oudenaarden A., Jaenisch R., Linhart H.G. Genes methylated by DNA methyltransferase 3b are similar in mouse intestine and human colon cancer. J. Clin. Invest. 2011;121:1748–1752. doi: 10.1172/JCI43169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tao Y., Kang B., Petkovich D.A., Bhandari Y.R., In J., Stein-O'brien G., Kong X., Xie W., Zachos N., Maegawa S. Aging-like spontaneous epigenetic silencing facilitates Wnt activation, stemness, and Braf(V600E)-induced tumorigenesis. Cancer Cell. 2019;35:315–328.e6. doi: 10.1016/j.ccell.2019.01.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ushijima T., Suzuki H. The origin of CIMP, at last. Cancer Cell. 2019;35:165–167. doi: 10.1016/j.ccell.2019.01.015. [DOI] [PubMed] [Google Scholar]
  48. Vaughn C.P., Zobell S.D., Furtado L.V., Baker C.L., Samowitz W.S. Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer. Genes Chromosomes Cancer. 2011;50:307–312. doi: 10.1002/gcc.20854. [DOI] [PubMed] [Google Scholar]
  49. Weisenberger D.J., Siegmund K.D., Campan M., Young J., Long T.I., Faasse M.A., Kang G.H., Widschwendter M., Weener D., Buchanan D. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat. Genet. 2006;38:787–793. doi: 10.1038/ng1834. [DOI] [PubMed] [Google Scholar]
  50. Xue W., Zender L., Miething C., Dickins R.A., Hernando E., Krizhanovsky V., Cordon-Cardo C., Lowe S.W. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature. 2007;445:656–660. doi: 10.1038/nature05529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yaeger R., Chatila W.K., Lipsyc M.D., Hechtman J.F., Cercek A., Sanchez-Vega F., Jayakumaran G., Middha S., Zehir A., Donoghue M.T.A. Clinical sequencing defines the genomic landscape of metastatic colorectal cancer. Cancer Cell. 2018;33:125–136.e3. doi: 10.1016/j.ccell.2017.12.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yaeger R., Corcoran R.B. Targeting alterations in the RAF-MEK pathway. Cancer Discov. 2019;9:329–341. doi: 10.1158/2159-8290.CD-18-1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yang S., Farraye F.A., Mack C., Posnik O., O'brien M.J. BRAF and KRAS Mutations in hyperplastic polyps and serrated adenomas of the colorectum: relationship to histology and CpG island methylation status. Am. J. Surg. Pathol. 2004;28:1452–1459. doi: 10.1097/01.pas.0000141404.56839.6a. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods and Figures S1–S5
mmc1.pdf (3.4MB, pdf)

Data Availability Statement

RNA-seq and DNA methylation data is available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126434. The accession number for the data reported in this paper is GSE126434: secure token for access exqpcwyshxsfdgd).

Articles from iScience are provided here courtesy of Elsevier

RESOURCES